Journal: Cellular and Molecular Life Sciences
Article Title: Intracellular FGF1 protects cells from apoptosis through direct interaction with p53
doi: 10.1007/s00018-023-04964-9
Figure Lengend Snippet: FGF1 interacts directly with p53. a Western blot analysis of pull-down experiment using U2OS cell lysate and recombinant SBP-FGF1. Cell lysates were incubated with SBP-FGF1 immobilized on Streptavidin-Agarose resin or with Streptavidin-Agarose resin alone for 1 h, then the resins were washed and proteins in the complex were analyzed using anti-p53 and anti-FGF1 antibodies. b Western blot analysis of pull-down experiment using U2OS stably transfected with SBP-FGF1_pcDNA3.1 (SBP-FGF1) or empty pcDNA3.1 (–) vectors. Cell lysates were incubated with Streptavidin-Agarose resin alone for 1 h, then the resins were washed and proteins in the complex were analyzed using anti-p53 and anti-FGF1 antibodies. c Proximity ligation assay in U2OS cells stably transfected with myc-FGF1_pcDNA3.1 (FGF1) or empty pcDNA3.1 (–) vectors showing the complexes of FGF1 and endogenous p53. Cells were fixed with 4% paraformaldehyde and subjected to the in situ PLA procedure using goat anti-FGF1 and mouse anti-p53 antibodies. A confocal z-stack including a whole cell was performed to observe the maximum amount of PLA signals. Cell nuclei were counterstained with DAPI. Representative images and quantification of PLA puncta per nucleus are shown. 41 images of each sample were analyzed. The box-and-whiskers graphs show the median, the 25th and 75th percentiles (box), and the 90th and 10th percentiles (whiskers). Statistical significance: ***p < 0.001. d FGF1 binds to p53 within its DNA-binding domain. Western blot analysis of pull-down experiment using recombinant His-tagged full-length p53 and its DNA-binding domain (p53_DBD). p53 proteins were incubated with recombinant SBP-FGF1 immobilized on Streptavidin-Agarose resin or Streptavidin-Agarose resin alone for 1 h, then the resins were washed and proteins in the complex were analyzed using anti-His-tag and anti-FGF1 antibodies. e Kinetics of p53: FGF1 and p53_DBD: FGF1 interaction assessed with SPR. The FGF1 protein at the concentrations from 0.016 μM to 2.048 μM was injected on CM5 sensor surface with p53 or DNA-binding domain of p53 immobilized at 1000 RU or 800 RU, respectively. Equilibrium dissociation constant (K D ) was calculated from saturation binding curve
Article Snippet: The recombinant human full-length p53 and its DNA-binding domain (p53_DBD) dissolved in 10 mM sodium acetate, pH 5.0 were immobilized on CM5 sensor chip surface (GE Healthcare) at about 1000 RU and 800 RU, respectively, using an amine coupling protocol.
Techniques: Western Blot, Recombinant, Incubation, Stable Transfection, Transfection, Proximity Ligation Assay, In Situ, Binding Assay, Injection
